Higher gamma-glutamyl transferase levels are associated with an increased risk of incident systemic sclerosis: a nationwide population-based study

Gamma-glutamyl transferase (GGT) is known to promote oxidative stress. As oxidative stress is a key component in the pathogenesis of systemic sclerosis (SSc), we investigated whether GGT levels are associated with the risk of incident SSc. A cohort of individuals without SSc who underwent national health examination in 2009 were extracted from the Korean National Health Insurance Service database. The incidence rate of SSc during the observation period, between 2009 and 2019, was estimated. GGT levels measured in 2009 were categorized into quartiles (Q1 [lowest], Q2, Q3, and Q4 [highest]). Multivariable Cox proportional hazard models were used to estimate the risk of incident SSc according to the quartiles of GGT, using Q1 as the reference. A total of 6,091,788 individuals were included. Incidence rate of SSc was 1.16 per 100,000 person-years over a mean observation period of 9.2 years. After adjusting for age, sex, body mass index, economic income, smoking status, alcohol consumption, physical activity, hypertension, type 2 diabetes, dyslipidemia, and chronic kidney disease, higher quartiles of GGT levels were significantly associated with a higher risk of incident SSc (Q4: adjusted hazard ratio [aHR] 1.807, 95% confidence interval CI 1.446–2.259; Q3: aHR 1.221, 95% CI 0.971–1.536; and Q2: aHR 1.034, 95% CI 0.807–1.324; p for trend < 0.001). Higher GGT levels were associated with a higher risk of incident SSc. These findings could lead to a closer monitoring for high risk individuals and an earlier diagnosis and treatment.


Definitions of outcome and covariates
The study outcome was the incidence of SSc, which was defined as a rare intractable disease (RID) with a code of V138 23 .In the Korean RID system, the RID code is given after a thorough review of the fulfillment of the diagnostic criteria provided by the NHIS 22 .Covariates including smoking status, alcohol consumption, and physical activity, were defined based on data from standardized self-reporting questionnaires, which are included in the national health check-up data.According to the questionnaires, smoking status was categorized into nonsmoker (who never smoked), ex-smoker (who previously had smoked but not currently), and current smoker (who smokes currently).Alcohol consumption was categorized into non-drinker (0 g/day), mild drinker (> 0 g/ day and < 30 g/day), and heavy drinker (≥ 30 g/days).Regarding physical activity, regular exercise was defined as a moderate exercise ≥ 5 days or vigorous exercise ≥ 3 days per week 22 .Other covariates were defined based on ICD-10 codes, prescription, laboratory data, and BP as described in previous studies (Supplemental Table 1) 24 .

Statistical analysis
Continuous variables that followed normal distribution were expressed as mean ± standard deviation, and continuous variables that followed skewed distribution were Log-transformed and expressed as geometric mean (95% confidence interval [CI]).The normality of the continuous variables was tested using Kolmogorov-Smirnov test and histogram.Categorical variables are expressed as numbers (%).Continuous variables were compared using one-way analysis of variance, and categorical variables were compared using Chi 2 test.The incidence rate of SSc was calculated as the number of events per 100,000 person-years.Cox proportional hazard analyses were used to investigate the association between baseline GGT levels and incidence of SSc.Using Q1 levels of GGT as the reference, hazard ratios (HRs) and 95% CIs according to the quartiles of GGT levels were estimated.Model 1 was a crude model.Model 2 was adjusted for age and sex.Model 3 was adjusted for age, sex, BMI, income, smoking status, alcohol consumption, and physical activity.Model 4 was additionally adjusted for hypertension, type 2 diabetes, dyslipidemia, and chronic kidney disease (CKD).As a sensitivity analysis, we categorized the levels of GGT to deciles (D1 [lowed 10%] to D10 [highest 10%]) and analyzed the risk of incident SSc according to the deciles of GGT levels, using D1 as the reference.Subgroup analysis was performed to assess whether there is a subset of individuals in whom the association between GGT levels and risk of incident SSc is more pronounced.All p-values were two-sided, and a p-value < 0.05 was considered statistically significant.Statistical analyses were performed using SAS version 9.4 (SAS Institute, Cary, NC, USA).

Ethics approval and consent to participate
This study was approved by the Institutional Review Board (IRB) of Gangnam Severance Hospital (No: 3-2022-0338).Owing to the retrospective nature of this study, the requirement for informed consent was waived by the IRB of Gangnam Severance Hospital.This study conformed the ethical guidelines laid out by the 1964 Helsinki declaration.

Baseline characteristics
Of the total 6,091,788 individuals included in this study, 654 individuals developed SSc during a mean follow-up of 9.2 ± 1.1 years, accounting for an incidence rate of 1.16 per 100,000 person-years.The comparison of baseline characteristics between individuals with different quartiles of GGT levels is shown in Table 1.Individuals with higher quartiles of GGT levels were older, had a higher BMI, more commonly had a low income (lowest 25%), were more commonly current smokers, heavy alcohol drinkers, less commonly performed regular physical activity, more commonly had hypertension, type 2 diabetes, dyslipidemia, and CKD, had higher systolic BP, diastolic BP, fasting glucose, total cholesterol, low-density lipoprotein cholesterol, triglyceride, AST, and ALT levels, and had lower high-density lipoprotein cholesterol levels and estimated glomerular filtration rates (all p for trend < 0.001).The geometric mean values (95% CI) of GGT in individuals with GGT in Q1, Q2, Q3, and Q4 were 12.75 (12.

Risk of incident SSc according to the GGT levels
The incidence rate and risk of incident SSc according to the GGT levels are reported in Table 2.The incidence rates of SSc in individuals with GGT levels in Q1, Q2, Q3, and Q4 were 0.95, 0.91, 1.14, and 1.64 per 100,000 person-years, respectively.In the crude model (model 1), individuals with higher quartile levels of GGT had a higher risk of incident SSc than those with Q1 levels of GGT (Q4: unadjusted HR 1.723, 95% CI 1.395-2.127;In the sensitivity analysis (Table 3) where GGT levels were categorized into deciles to test the robustness of the association between higher GGT levels and higher risk of incident SSc, similar associations were observed as in the quartile-based analysis.

Subgroup analysis
For subgroup analysis, individuals were stratified according to multiple covariates.The results of the subgroup analysis are summarized in Table 4.The association between higher GGT levels (Q4 compared with Q1-Q3) and increased risk of incident SSc was more pronounced in males than in females (adjusted HR 2.483 vs. 1.504, p for interaction = 0.016).

Discussion
In this large-scale cohort study, we found that higher GGT levels are associated with an increased risk of incident SSc in the general population.This association was consistently observed when GGT levels were categorized in both quartiles and deciles, thus adding robustness to our results.In addition, we found that this association was stronger in males than in females.To our knowledge, this is the first study to assess the association between GGT levels and risk of incident SSc.Beyond its traditional use as a marker for alcohol-related liver diseases and hepatobiliary diseases 19 , considering its enzymatic role in promoting oxidative stress 20,21 , GGT is now considered a marker for various diseases such as metabolic syndrome, atherosclerosis, arterial plaque, heart failure, diabetes, and several types of cancers 25 .Our data add to growing knowledge that GGT could be considered as a predictive marker of SSc.Considering that GGT promotes oxidative stress 20,21 , and that oxidative stress is an important factor involved in the pathogenesis   www.nature.com/scientificreports/ of SSc 4 , the association between GGT levels and the risk of incident SSc observed in our study is convincing.In addition, independent of its enzymatic activity, GGT upregulates the gene expression of tissue factor (TF) in human peripheral blood mononuclear cells, and increases TF-related pro-coagulant activity 26 .As TF-thrombin signaling enhances the fibrotic activity of myofibroblasts in SSc 27 , the upregulation of TF expression by GGT may also explain the association between GGT levels and the risk of incident SSc.
In the subgroup analysis, we found that the association between higher GGT levels and increased risk of incident SSc was more pronounced in males than in females.In other words, the association between higher GGT levels and increased risk of incident SSc was weaker in females.As females per se pose a higher risk of incident SSc than males 28 , the influence of GGT on incidence of SSc may be attenuated in females, resulting in a less prominent association between higher GGT levels and increased risk of incident SSc in females.
A number of studies have evaluated the risk factors of incident SSc: middle age (45-64 years) and female sex have been consistently reported as risk factors of incident SSc 28 .In the multivariable models (model 2-4) of our study, age and sex were adjusted and the association between higher quartiles of GGT levels and risk of incident SSc was observed consistently throughout the models.These indicate that GGT levels could be considered as a risk factor of incident SSc, independent of age and sex.
The upper normal limit of GGT level in Korea is 63 IU/L for males, and 35 IU/L for females 29,30 .In our study, the cut-off of Q4 levels of GGT was ≥ 49 IU/L for males, and ≥ 22 IU/L for females, which are lower than the upper normal limits.As the statistically significant association between quartiles of GGT levels and higher risk of incident SSc was observed in the comparison of Q4 vs. Q1 (model 4, adjusted HR 2.038, 95% CI 1.733-2.396)(Table 2), it is important to note that higher levels of GGT are associated with an increased risk of incident SSc, even when the GGT levels are within the upper normal limit.When analyzed in deciles, the cut-offs of D10 levels of GGT were ≥ 82 IU/L for males, and ≥ 33 IU/L for females.That is, the cut-off of D10 levels for males was above the upper normal limit (63 IU/L), and the cut-off of D10 levels for females was approximately the same as the upper normal limit (35 IU/L).The effect size became exponentially larger in the comparison of D10 vs. D1 (model 4, adjusted HR 2.215) compared with the comparison of D9 vs. D1 (model 4, adjusted HR 1.310) (Table 3).Taken together, the risk of incident SSc increases as the GGT level rises within the upper normal limit, and increases overwhelmingly when the GGT level rises above the upper normal limit.
There are few limitations of this study.First, as this was a Korean population-based study, the results of our study may not be generalizable to other ethnic populations.Second, as the data regarding the phenotypes of SSc, such as organ involvement pattern and modified Rodnan skin score, are unavailable in the NHIS database, we could not analyze the association between GGT levels and SSc phenotypes.Third, as this was a retrospective study, causality could not be fully explained.However, we did apply a 1-year lag period for a better causality assessment.Fourth, as this was a nationwide study, interlaboratory variation on GGT levels may exist.However, previous study has reported that the interlaboratory coefficient of variation for GGT is 11.6%, which is an acceptable level 31 .

Conclusions
In conclusion, higher GGT levels were independently associated with an increased risk of incident SSc.The effect size increased gradually as the GGT level rose within the upper normal limit, and exponentially increased when the GGT level rose above the upper normal limit (Fig. 2).Regardless of whether the GGT level was above the upper normal limit or not, the effect size increased significantly as the GGT level rose.Our data suggest that individuals with higher levels of GGT could be considered as having an increased risk of developing SSc.Thus, the clinicians should be aware that close monitoring for the development of SSc is warranted in individuals with higher GGT levels.This could eventually lead to an early diagnosis and treatment of SSc.

Figure 1 .
Figure 1.Selection of the study population from the NHIS database.NHIS National Health Insurance Service, SSc systemic sclerosis.

Figure 2 .
Figure 2. Summary of the present study.GGT gamma-glutamyl transferase, NHIS National Health Insurance Service, HR hazard ratio, CI confidence interval.

Table 1 .
Comparison of baseline characteristics according to the baseline quartiles of GGT levels.ALT alanine aminotransferase, AST aspartate aminotransferase, BMI body mass index, BP blood pressure, CKD chronic kidney disease, eGFR estimated glomerular filtration rate, GGT gamma-glutamyl transferase, HDL-C highdensity lipoprotein cholesterol, LDL-C low-density lipoprotein cholesterol.a P for trend.b Geometric mean (95% confidence interval).

Table 2 .
Incidence of systemic sclerosis according to quartiles of GGT at baseline.Model 1: Crude model.Model 2: Adjusted for age and sex.Model 3: Adjusted for age, sex, BMI, income, smoking status, alcohol consumption, and physical activity.Model 4: Adjusted for age, sex, BMI, income, smoking status, alcohol consumption, physical activity, hypertension, type 2 diabetes, dyslipidemia, and CKD.P for trend < 0.001 in all models.BMI body mass index, CI confidence interval, CKD chronic kidney disease, GGT gamma-glutamyl transferase, HR hazard ratio, IR incidence ratio, pyrs person-years.

Table 3 .
Incidence of systemic sclerosis according to deciles of GGT at baseline.Model 1: Crude model.Model 2: Adjusted for age and sex.Model 3: Adjusted for age, sex, BMI, income, smoking status, alcohol consumption, and physical activity.Model 4: Adjusted for age, sex, BMI, income, smoking status, alcohol consumption, physical activity, hypertension, type 2 diabetes, dyslipidemia, and CKD.P for trend < 0.001 in all models.BMI body mass index, CI confidence interval, CKD chronic kidney disease, GGT gamma-glutamyl transferase, HR hazard ratio, IR incidence ratio, pyrs person-years.

Table 4 .
Subgroup analysis for the association between levels of GGT and incidence of systemic sclerosis.